Project Summary/Abstract Spatio-temporal regulation of exocytosis is essential for establishing and maintaining intercellular junctions and surface polarity, properties vital to epithelial tissues. Failure to accurately deliver and maintain correct levels of membrane proteins at appropriate sites is a common feature of human metabolic diseases and cancers. Despite progress in understanding mechanisms of protein sorting, trafficking and turnover, major questions persist about these processes. The long-term goal of our research is to understand how environmental cues signal to the cytoskeleton and transport machinery to organize trafficking pathways in epithelial cells. The focus of our research is on the exocyst, a multi-functional scaffold, and Ral GTPases, which regulate exocyst activities. Based upon results of our previous work, we have developed a working hypothesis. We propose that RalA and RalB are coordinately regulated by cell-cell adhesion, and that they control distinct stages of exocyst-dependent membrane trafficking in both the exocytic and endocytic pathways by controlling the association between the scaffold and additional proteins. We have identified two such proteins. Munc18c, a regulator of vesicle fusion, binds the exocyst in a RalA-dependent manner and this interaction is required for basolateral exocytosis. USP9X, a deubiquitinase, engages the exocyst in a RalB-dependent manner and stabilizes proteins in the plasma membrane during intercellular junction assembly. Therefore, we propose that coordinated regulation of a common effector complex (exocyst) by two related GTPases (RalA and RalB) provides a key molecular link between an environmental cue (cell-cell adhesion) and biogenesis of epithelial cell specific structures. The overall objective of this proposal is to dissect molecular mechanisms by which Ral GTPases and exocyst complexes control the formation of intercellular junctions and polarized membrane domains in epithelial cells. The specific aims of this study are: 1) to identify mechanisms by which E-cadherin- mediated cell adhesion regulates Ral GTPase activities; 2) to determine how RalA engages exocyst complexes to facilitate basolateral exocytosis; and 3) to define how RalB engages the exocyst to regulate endocytosis of components of apical junctional complexes. Collectively, the studies we propose here seek to uncover important details of a mechanism that links cell-cell adhesion to activation of a signaling pathway that promotes polarized membrane trafficking and selective protein stabilization during establishment of epithelial polarity. Given that the molecules on which we will focus are conserved across metazoans, we believe that studying how they collaborate to establish and maintain epithelial structures will provide important insights into mechanisms that regulate assembly of plasma membrane protein complexes in many other cell types and organisms. Moreover, because Ral GTPases and exocyst components are involved in many cancers, metabolic disorders and diabetes, the significance of this work is that it will guide our thinking about new therapies for human diseases in which these factors are disrupted.